Project summary. The innate immune and inflammatory response to influenza A virus is a major contributor to influenza disease, because it promotes viral pneumonia and secondary infections through lung damage. As a consequence, it has recently become clear that blocking influenza virus replication is not sufficient to treat influenza disease. New strategies to block the innate immune and inflammatory responses need to be developed. These strategies could take advantage of the role of viral proteins in modulating host responses, including the host shutoff factor PA-X, which targets host RNAs for degradation. PA-X inhibits immune responses, and viruses that lack PA-X trigger stronger cytokine responses in mouse and chicken infection models. However, these stronger responses do not promote clearance, but in many cases result in increased morbidity and mortality. This indicates that PA-X has a different function from other influenza immune modulators, and that PA-X can be protective for the host. Defining the mechanism of action of PA-X will be important to understand the unique contribution of PA-X to virus-host interplay and to exploit it to modulate host responses to influenza. While host shutoff factors are usually considered indiscriminate, we have found that PA-X is selective, and targets specific types of RNAs. Particularly, we have uncovered a connection between PA-X and splicing of host RNAs. We have found that PA-X preferentially degrades spliced RNAs in infected cells and, through proximity-dependent protein labeling, we have identified candidate interaction partners of PA-X, which are cellular proteins involved in RNA splicing and 3' end processing. These data have led us to the central hypothesis of this project: PA-X selectively targets RNAs through interactions with cellular splicing and RNA processing machinery. The connection between splicing and PA-X activity is novel among host shutoff RNases. It also suggests that through interactions with splicing factors, PA-X may differentially regulate host RNAs with important functions in immune and inflammatory responses, including spliced vs. intronless types of interferons, key antiviral cytokines. Our objective is to examine the connection between PA-X and cellular mRNA splicing and its effects on cytokine regulation. The long-term goal of our research is to link the molecular function of PA-X to its role in vivo. In Aim 1, we will determine how the number of splice sites and their position affects PA-X's ability to target RNAs, and how widely applicable this is to PA-X variants from divergent influenza strains. We will use a combination of reporter assays and high-throughout RNAseq. In Aim 2, we will define the role of candidate cellular co-factors of PA-X in RNA targeting by PA-X, particularly association with target RNAs and subcellular localization of PA-X using knock-down approaches. We will then use viral mutants to test the role of these interactions in mouse infections. We expect that these experiments will define RNA characteristics and proteins that are important for PA-X activity, expanding our understanding of influenza-host interplay and pointing to strategies to alter PA-X activity for immune modulation.